This proposal is focused on the characterization of transport systems in R. prowazekii, the etiologic agent of epidemic typhus. R. prowazekii has only 834 genes. Such a small genome size is possible because many intermediates required for their metabolism need not be synthesized, but are transported from the cytoplasm of the host cell by unusual transport systems that often have no counterparts in free-living bacteria. Aim I. Structure and function of the ATP/ADP transport system. Using cysteine scanning mutagenesis and the derivatization and cross-linking of cysteine residues, we will characterize essential residues and the packing of transmembrane helices that configure the formation of aqueous channels and active sites. Our biodiversity approach will be extended and a new effort to select for the TLC system in E. coli will be initiated. Aim II. Characterization of transport systems with multiple homologous genes. II.A. Functions of the four tlc genes paralogous to that for ATP/ADP transport. II.B. Function of the seven paralogous proline transport genes. We will determine the nature of the transport systems encoded by these twelve paralogous rickettsial genes when cloned into E. coli and in native R. prowazekii. We will establish their natural substrates in rickettsiae, their energy coupling, and by using knock-out recombinants determine whether all of these twelve paralogs are essential. Aim III. ABC-transport systems in rickettsiae: focused on glutamine transport. Two hallmarks of the ABC-transport system are remarkable high affinity and the ability to establish a very high thermodynamic transmembrane gradient of substrate. Such a transport system seems inappropriate in R. prowazekii living in cytoplasm. We will focus on the glutamine transport system. The alternative hypotheses that we will investigate are: 1) the rickettsial glutamine-ABC is high affinity, always works at saturation levels of host cytoplasmic glutamine, and does not care about inefficiency because there is plenty of ATP around; 2) high affinity of this transport system is appropriate because, although the total concentration of glutamine in host cytoplasm is high, the free concentration available to the rickettsia is low because of competition with tRNA; 3) these genes in R. prowazekii are not used to transport glutamine but are in the process of mutational melt-down; and 4) the rickettsial glutamine transport system is now non-functional, with substrate recognition residing totally in the membrane protein.